Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (μUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-μUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for μUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed μUtrn. As such, we found that μUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike μUtrn, μdystrophin (μDys) expression did not show this fiber type preference. Interestingly, μUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & μUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.

Dynamical signatures of isometric force control as a function of age, expertise, and task constraints

From the conceptual and methodological framework of the dynamical systems approach, force control results from complex interactions of various subsystems yielding observable behavioral fluctuations, which comprise both deterministic (predictable) and stochastic (noise-like) dynamical components. Here, we investigated these components contributing to the observed variability in force control in groups of participants differing in age and expertise level. To this aim, young (18–25 yr) as well as late middle-aged (55−65 yr) novices and experts (precision mechanics) performed a force maintenance and a force modulation task. Results showed that whereas the amplitude of force variability did not differ across groups in the maintenance tasks, in the modulation task it was higher for late middle-aged novices than for experts and higher for both these groups than for young participants. Within both tasks and for all groups, stochastic fluctuations were lowest where the deterministic influence was smallest. However, although all groups showed similar dynamics underlying force control in the maintenance task, a group effect was found for deterministic and stochastic fluctuations in the modulation task. The latter findings imply that both components were involved in the observed group differences in the variability of force fluctuations in the modulation task. These findings suggest that between groups the general characteristics of the dynamics do not differ in either task and that force control is more affected by age than by expertise. However, expertise seems to counteract some of the age effects. NEW & NOTEWORTHY Stochastic and deterministic dynamical components contribute to force production. Dynamical signatures differ between force maintenance and cyclic force modulation tasks but hardly between age and expertise groups. Differences in both stochastic and deterministic components are associated with group differences in behavioral variability, and observed behavioral variability is more strongly task dependent than person dependent.

Active shortening protects against stretch-induced force deficits in human skeletal muscle

Skeletal muscle contraction results from molecular interactions of myosin “crossbridges” with adjacent actin filament binding sites. The binding of myosin to actin can be “weak” or “strong,” and only strong binding states contribute to force production. During active shortening, the number of strongly bound crossbridges declines with increasing shortening velocity. Forcibly stretching a muscle that is actively shortening at high velocity results in no apparent negative consequences, whereas stretch of an isometrically (fixed-length) contracting muscle causes ultrastructural damage and a decline in force-generating capability. Our working hypothesis is that stretch-induced damage is uniquely attributable to the population of crossbridges that are strongly bound. We tested the hypothesis that stretch-induced force deficits decline as the prevailing shortening velocity is increased. Experiments were performed on permeabilized segments of individual skeletal muscle fibers obtained from human subjects. Fibers were maximally activated and allowed either to generate maximum isometric force (Fo), or to shorten at velocities that resulted in force maintenance of ≈50% Fo or ≈2% Fo. For each test condition, a rapid stretch equivalent to 0.1 × optimal fiber length was applied. Relative to prestretch Fo, force deficits resulting from stretches applied during force maintenance of 100, ≈50, and ≈2% Fo were 23.2 ± 8.6, 7.8 ± 4.2, and 0.3 ± 3.3%, respectively (means ± SD, n = 20). We conclude that stretch-induced damage declines with increasing shortening velocity, consistent with the working hypothesis that the fraction of strongly bound crossbridges is a causative factor in the susceptibility of skeletal muscle to stretch-induced damage. NEW & NOTEWORTHY Force deficits caused by stretch of contracting muscle are most severe when the stretch is applied during an isometric contraction, but prevented if the muscle is shortening at high velocity when the stretch occurs. This study indicates that velocity-controlled modulation of the number of strongly bound crossbridges is the basis for the observed relationship between stretch-induced muscle damage and prevailing shortening velocity.

Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.

Mechanism of Catch Force: Tethering of Thick and Thin Filaments by Twitchin

Catch is a mechanical state occurring in some invertebrate smooth muscles characterized by high force maintenance and resistance to stretch during extremely slow relaxation. During catch, intracellular calcium is near basal concentration and myosin crossbridge cyctng rate is extremely slow. Catch force is relaxed by a protein kinase A-mediated phosphorylation of sites near the N- and C- temini of the minititin twitchin (~526 kDa). Some catch force maintenance car also occur together with cycling myosin crossbridges at submaximal calcium concentrations, but not when the muscle is maximally activated. Additionally, the link responsible for catch can adjust during shortening of submaximally activated muscles and maintain catch force at the new shorter length. Twitchin binds to both thick and thin filaments, and the thin filament binding shown by both the N- and Cterminal portions of twitchin is decreased by phosphorylation of the sites that regulate catch. The data suggest that the twitchin molecule itself is the catch force beanng tether between thick and thin filaments. We present a model for the regulation of catch in which the twitchin tether can be displaced from thin filaments by both (a) the phosphorylation of twitchin and (b) the attachment of high force myosin crossbridges.